Ferredoxin Reductase Research Articles

Biological effects of radiation exposure in patients treated with X-ray guided endovascular aortic repair

Abstract Background X-ray guided cardiovascular interventions such as endovascular aortic repair (EVAR) expose patients and operators to significant amounts of repeated ionising radiation over a long period of time. The biological effect of such protracted radiation exposure is poorly understood. We have previously demonstrated a higher incidence of dicentric chromosome (DC) aberrations in high-volume endovascular interventionalists and differences in acute DNA damage in lymphocytes between individuals following in vitro irradiation. Studies suggested a higher incidence of cancer in patients who underwent EVAR compared with those who have had open aneurysm repair, but the evidence is inconclusive. Further work is required to evaluate the biological radiation risk of X-ray guided cardiovascular interventions. Purposes We aimed to examine radiation-induced genome instability from patients after complex EVAR. We also aimed to investigate the intrinsic response of each patient to radiation using biomarkers of acute DNA damage following in vitro irradiation of their blood and to correlate this response to the expression of radiation-responsive genes. Methods Lymphocytes were isolated from patients after complex (branched/fenestrated) EVAR and non-irradiated controls. DC, a type of chromosomal aberration caused by radiation exposure were enumerated. γ-H2AX, a marker of acute DNA damage/repair, was measured by immunofluorescence after in vitro irradiation at 0.2 Gy and 1 Gy, along with the expression of the radiation-responsive genes FDXR, CCNG1, P21 and PHPT1 using qPCR. Results 17 patients (82% male, median age 73[59 – 85years]) and 16 controls (56% male, median age 68[53 – 83years]) were recruited. The mean incidence of DC was 3.782 (95% CI 3.13 - 4.43) and 0.898 (95% CI 0.48 -1.32) per 1000 cells for patients and controls, respectively (P<0.0001). Patients had higher background of γ-H2AX foci than unexposed controls, (0.7056, 95% CI 0.316 - 1.10) vs (0.241 95% CI 0.077 - 0.405) per cell, P<0.05. FDXR expression was most increased following irradiation (P<0.0001), reaching a mean 13.1-fold and 11.4-fold increase at 4-hour and 24-hour time-point, respectively, but this upregulation was not significantly different between the two groups. Conclusion We have shown an increased frequency of chromosomal aberrations in patients after complex EVAR, a biological finding that may support a propensity to developing malignancies. Patients also have higher basal DNA damage/repair (γ-H2AX) activity. Expression of ferredoxin reductase, FDXR gene could be a potential radio-responsive biomarker to provide personalised biological dosimetry information for clinical monitoring. Our study further underlines the long-term radiation risk of X-ray guided cardiovascular interventions and the potential of biological assays that may guide personalised care.

Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2.

Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL) is a rare, orphan autosomal recessive disorder caused by mutations in ferredoxin-2 (FDX2), which is a [2Fe-2S] cluster-binding protein participating in the formation of iron-sulfur clusters in mitochondria. In this biosynthetic pathway, FDX2 works as electron donor to promote the assembly of both [2Fe-2S] and [4Fe-4S] clusters. A recently identified missense mutation of MEOAL is the homozygous mutation c.431C>T (p.P144L) described in six patients from two unrelated families. This mutation alters a highly conserved proline residue located in a loop of FDX2 that is distant from the [2Fe-2S] cluster. How this Pro to Leu substitution damages iron-sulfur cluster biosynthesis is unknown. In this work, we have first compared the structural, dynamic, cluster binding and redox properties of WT and P144L [2Fe-2S] FDX2 to have clues on how the pathogenic P144L mutation can perturb the FDX2 function. Then, we have investigated the interaction of both WT and P144L [2Fe-2S] FDX2 with its physiological electron donor, ferredoxin reductase FDXR, comparing their electron transfer efficiency and protein-protein recognition patterns. Overall, the data indicate that the pathogenic P144L mutation negatively affects the FDXR-dependent electron transfer pathway from NADPH to FDX2, thereby reducing the capacity of FDX2 in assembling both [2Fe-2S] and [4Fe-4S] clusters. Our study also provided solid molecular evidences on the functional role of the C-terminal tail of FDX2 in the electron transfer between FDX2 and FDXR.

FDXR gene expression and micronucleus frequency in Type 2 diabetes mellitus patients and their importance in case of radiation exposures

Analysis of gene expression (e.g. Ferredoxin Reductase: FDXR) changes in the blood samples have shown potential as predictive markers of disease, prognosis for therapy as well as triage and dose estimation in radiation-exposed populations. Similarly, quantification of micronuclei (MN) formation has been presented as a rapid cytogenetic marker for those applications. It was cautioned that the reliable utilization of these markers for prediction of disease, therapy prognosis, and dose estimation depends upon the information on known variables that affect these markers. Literature suggests that advanced glycation end products/oxidative stress in diabetic conditions can alter the levels of DNA damage and gene expression, and impact the segregation of the exposed from unexposed during nuclear disasters. Therefore, we investigated the influence of Type 2 diabetes mellitus (T2DM) on baseline expression of the FDXR and frequency of MN. Peripheral blood samples were collected from healthy volunteers (HV) (n = 60; 43 males and 17 females) and T2DM patients (n = 60; 32 males and 28 females), and performed real-time quantification of FDXR gene expression and analysis of MN frequency using microscopy. The basal level of FDXR gene expression (2.55 folds) (p < 0.01) and the frequency of MN is significantly (p < 0.01) higher (4 folds) in T2DM patients when compared to HV. Further, subgroup analysis found that gender, alcohol, smoking, duration of T2DM, complications, and medications increased both the expression of the FDXR gene and frequency of MN in T2DM; nevertheless, the increase was not significant, except for gender (p < 0.05) and medication (p < 0.05) on the frequency of MN. Overall results indicate that the T2DM patients showed a higher basal level expression of the FDXR gene and MN frequency when compared to HV and suggest an altered metabolic condition in T2DM is a confounding factor that impacts the levels of those markers. The increased levels of these markers might need to be considered to monitor medical radiation exposures and reliable biodosimetry during large-scale radiological accidents.

Identification of Electron Transfer in the System of Ferredoxins and Ferredoxin Reductases from Mycolicibacterium smegmatis

Steroid-26-monooxygenases belong to the cytochrome P450 superfamily and function as part of three-component systems together with ferredoxins and ferredoxin reductases providing electron transport. The P450-dependent redox partners of the actinobacterial strain Mycolicibacterium smegmatis mc2155 were investigated. The genes encoding mycolibacterial ferredoxins (FdxD and FdxE) and ferredoxin reductases (FdrA and FprA) were overexpressed in E. coli cells. A scheme for isolation and purification of synthesized recombinant proteins using affinity chromatography was developed, resulting in electrophoretically homogeneous preparations. Spectral analysis of ferredoxin reductase showed absorption peaks characteristic of FAD-containing proteins. The reaction of cytochrome c reduction using recombinant proteins was carried out, demonstrating that FdxD, FdxE, FdrA, and FprA can act as components of electron transport from the reducing equivalents of NAD(P)H.

Hormonal orchestra: mastering mitochondria's role in health and disease.

Mitochondria is a subcellular organelle involved in the pathogenesis of cellular stress, immune responses, differentiation, metabolic disorders, aging, and death by regulating process of fission, fusion, mitophagy, and transport. However, an increased interest in mitochondria as powerhouse for ATP production, the mechanisms of mitochondria-mediated cellular dysfunction in response to hormonal interaction remains unknown. Mitochondrial matrix contains chaperones and proteases that regulate intrinsic apoptosis pathway through pro-apoptotic Bcl-2 family's proteins Bax/Bak, and Cyt C release, and induces caspase-dependent and independent cells death. Energy and growth regulators such as thyroid hormones have profound effect on mitochondrial inner membrane protein and lipid compositions, ATP production by regulating oxidative phosphorylation system. Mitochondria contain cholesterol side-chain cleavage enzyme, P450scc, ferredoxin, and ferredoxin reductase providing an essential site for steroid hormones biosynthesis. In line with this, neurohormones such as oxytocin, vasopressin, and melatonin are correlated with mitochondrial integrity, displaying therapeutic implications for inflammatory and immune responses. Melatonin's also displayed protective role against oxidative stress and mitochondrial synthesis of ROS, suggesting a defense mechanism against aging-related diseases. An imbalance in mitochondrial bioenergetics can cause neurodegenerative disorders, cardiovascular diseases, and cancers. Hormone-induced PGC-1α stimulates mitochondrial biogenesis via activation of NRF1 and NRF2, which in turn triggers mtTFA in brown adipose and cardiac myocytes. Mitochondria can be transferred through cells merging, exosome-mediated transfer, and tunneling through nanotubes. By delineating the underlying molecular mechanism of hormonal mitochondrial interaction, this study reviews the dynamics mechanisms of mitochondria and its effects on cellular level, health, diseases, and therapeutic strategies targeting mitochondrial diseases.

Novel Variant of FDXR as a Molecular Etiology of Postlingual Post-Synaptic Auditory Neuropathy Spectrum Disorder via Mitochondrial Dysfunction: Reiteration of the Correlation between Genotype and Cochlear Implantation Outcomes.

FDXR encodes the mitochondrial ferredoxin reductase, which is associated with auditory neuropathy spectrum disorder (ANSD) and optic atrophy. Only two studies have described FDXRrelated hearing loss. The auditory rehabilitation outcomes of this disease entity have not been investigated, and the pathophysiologic mechanism is not well elucidated. Here we report a hearingimpaired subject with co-segregation of the FDXR variant and post-synaptic type ANSD, who underwent cochlear implantation (CI) with favorable outcomes. We suggest a possible pathophysiologic mechanism of adult-onset ANSD via mitochondrial dysfunction. A 35-year-old woman was ascertained to have ANSD. Exome sequencing identified the genetic cause of hearing loss, and functional study measuring mitochondrial activity was performed to provide molecular evidence of pathophysiology. Expression of FDXR in the mouse cochlea was evaluated by immunohistochemistry. Intraoperatively, electrically-evoked compound action potential (ECAP) responses were measured, and mapping parameters were adjusted accordingly. Audiological outcomes were monitored for over 1 year. In lymphoblastoid cell lines (LCLs) carrying a novel FDXR variant, decreased ATP and MtMP levels and increased ROS levels were observed compared to control LCLs. These dysfunctions were restored by administering mitochondria isolated from umbilical cord mesenchymal stem cells, confirming the pathogenic potential of this variant via mitochondrial dysfunction. Partial ECAP responses during CI and FDXR expression in the mouse cochlea indicate that FDXR-related ANSD is postsynaptic. By increasing the pulse width during mapping, the patient's CI outcomes showed significant improvement over 1-year post-CI. Post-synaptic ANSD due to a novel FDXR variant linked to mitochondrial dysfunction was identified first in a Korean, and 1-year post CI outcomes were reported for the first time in the literature. Excellent audiologic results were obtained, and our results reiterate the correlation between genotype and CI outcomes in ANSD.

An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

Functional characterization of two reductase KshBs in Mycobacterium fortuitum and their applications to C9 non-hydroxylated steroid production

3-ketosteroid-9α-hydroxylase (KSH), a two-component monooxygenase consisting of a Rieske oxygenase KshA and a ferredoxin reductase KshB, is a crucial enzyme involved in C-9 hydroxylation and is indispensable in the microbial catabolism of sterols. However, the in vivo function of KshB remains unclear. In this study, two reductase subunits, KshB1 and KshB2 from Mycobacterium fortuitum ATCC 35855, were first characterized and then engineered in the strain MFΔkstD, which produces 9-OHAD (9α-hydroxy-4-androstene-3,17-dione), to construct the strains producing AD (4-androstene-3,17-dione). The transcriptional level and specific enzyme activity of KshB1 under phytosterols induction was significantly higher compared to KshB2. After knocking out kshB1, AD peaked at 1.77 g/L at 48 h, subsequently being fully converted to 9-OHAD. Furthermore, we developed the stable AD-producer MFΔkstDΔkshB by null mutation both kshB1 and kshB2, although no AD was detected by single knocking out kshB2. Through gene complementation and the bioconversion of phytosterols, KshB1 emerged as the primary reductase in the KSH system, with KshB2 serving a complementary role. The stable AD-producer MFΔkstDΔkshB could produce 5.18 g/L, 6.91 g/L, and 9.03 g/L AD from the transformation of 10 g/L, 15 g/L, and 20 g/L phytosterols, respectively. In conclusion, these findings highlight a new strategy for the metabolic engineering of Mycobacterium fortuitum, involving the inactivation of the reductase KshB to facilitate the production of C9 non-hydroxylated steroidal intermediates.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among these defects are recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-related mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, patients with FRM often experience worsening or demise following stress associated with infections. We investigated 2 female patients with FRM carrying the potentially novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from 3 patients with FRM fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

Functional analysis, diversity, and distribution of the ean cluster responsible for 17β-estradiol degradation in sphingomonads.

17β-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2.IMPORTANCESteroid estrogens have been detected in diverse environments, ranging from oceans and rivers to soils and groundwater, posing serious risks to both human health and ecological safety. The United States National Toxicology Program and the World Health Organization have both classified estrogens as Group 1 carcinogens. Several model organisms (proteobacteria) have established the 4,5-seco pathway for estrogen degradation. In this study, the newly isolated Sphingomonas colocasiae C3-2 could degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway. The novel gene cluster ean (including eanA, eanB1, eanC, and eanD) responsible for E2 degradation by the 4,5-seco pathway was identified; the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 through the 9,10-seco pathway. The TetR family transcriptional regulator EanR acts as a transcriptional repressor of the ean cluster. The cluster ean was also found to be present in other reported E2-degrading sphingomonads, indicating the ubiquity of the E2 metabolism in the environment.

Identification of Electron Transfer in the System of Ferredoxins and Ferredoxin Reductases from Mycolicibacterium smegmatis

Identification of Electron Transfer in the System of Ferredoxins and Ferredoxin Reductases from Mycolicibacterium smegmatis

Abstract 4464: Ferredoxin 1 is essential for embryonic development and lipid homeostasis

Abstract Mammalian ferredoxin 1 and 2 (FDX1/2) belong to an evolutionary conserved family of iron-sulfur cluster containing proteins and act as electron shutters between ferredoxin reductase (FDXR) and numerous proteins involved in critical biological pathways. FDX1 is involved in biogenesis of steroids and bile acids, Vitamin A/D metabolism, and lipoylation of tricarboxylic acid (TCA) cycle enzymes. FDX1 has been extensively characterized biochemically but its role in physiology and lipid metabolism has not been explored. In this study, we generated Fdx1-deficient mice and showed that knockout of both alleles of the Fdx1 gene led to embryonic lethality. We also showed that like Fdxr+/− mice, Fdx1+/− mice had a shorter life span and were prone to steatohepatitis. However, unlike Fdxr+/− mice, Fdx1+/− mice were not prone to spontaneous tumors. Additionally, we showed that FDX1 deficiency led to lipid droplet accumulation possibly via the ABCA1-SREBP1/2 pathway. Specifically, untargeted lipidomic analysis showed that FDX1 deficiency led to alterations in several classes of lipids, including cholesterol, triacylglycerides, acylcarnitines, ceramides, phospholipids and lysophospholipids. Taken together, our data indicate that FDX1 is essential for mammalian embryonic development and lipid homeostasis at both cellular and organismal levels. Citation Format: Jin Zhang, Sharkur Mohibi, Xinbin Chen, Vivian Perng, Yanhong Zhang. Ferredoxin 1 is essential for embryonic development and lipid homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4464.

Ferredoxin 1 is essential for embryonic development and lipid homeostasis.

Mammalian ferredoxin 1 and 2 (FDX1/2) belong to an evolutionary conserved family of iron-sulfur cluster containing proteins and act as electron shutters between ferredoxin reductase (FDXR) and numerous proteins involved in critical biological pathways. FDX1 is involved in biogenesis of steroids and bile acids, Vitamin A/D metabolism, and lipoylation of tricarboxylic acid (TCA) cycle enzymes. FDX1 has been extensively characterized biochemically but its role in physiology and lipid metabolism has not been explored. In this study, we generated Fdx1-deficient mice and showed that knockout of both alleles of the Fdx1 gene led to embryonic lethality. We also showed that like Fdxr+/-+/-, Fdx1+/-+/- had a shorter life span and were prone to steatohepatitis. However, unlike Fdxr+/-+/-, Fdx1+/-+/- were not prone to spontaneous tumors. Additionally, we showed that FDX1 deficiency led to lipid droplet accumulation possibly via the ABCA1-SREBP1/2 pathway. Specifically, untargeted lipidomic analysis showed that FDX1 deficiency led to alterations in several classes of lipids, including cholesterol, triacylglycerides, acylcarnitines, ceramides, phospholipids and lysophospholipids. Taken together, our data indicate that FDX1 is essential for mammalian embryonic development and lipid homeostasis at both cellular and organismal levels.

CXCL10 upregulation in radiation-exposed human peripheral blood mononuclear cells as a candidate biomarker for rapid triage after radiation exposure

Purpose In case of a nuclear accident, individuals with high-dose radiation exposure (>1–2 Gy) should be rapidly identified. While ferredoxin reductase (FDXR) was recently suggested as a radiation-responsive gene, the use of a single gene biomarker limits radiation dose assessment. To overcome this limitation, we sought to identify reliable radiation-responsive gene biomarkers. Materials and methods Peripheral blood mononuclear cells (PBMCs) were isolated from mice after total body irradiation, and gene expression was analyzed using a microarray approach to identify radiation-responsive genes. Results In light of the essential role of the immune response following radiation exposure, we selected several immune-related candidate genes upregulated by radiation exposure in both mouse and human PBMCs. In particular, the expression of ACOD1 and CXCL10 increased in a radiation dose-dependent manner, while remaining unchanged following lipopolysaccharide (LPS) stimulation in human PBMCs. The expression of both genes was further evaluated in the blood of cancer patients before and after radiotherapy. CXCL10 expression exhibited a distinct increase after radiotherapy and was positively correlated with FDXR expression. Conclusions CXCL10 expression in irradiated PBMCs represents a potential biomarker for radiation exposure.

Metal dyshomeostasis in the substantia nigra of patients with Parkinson's disease or multiple sclerosis.

Abnormal metal distribution in vulnerable brain regions is involved in the pathogenesis of most neurodegenerative diseases, suggesting common molecular mechanisms of metal dyshomeostasis. This study aimed to compare the intra- and extra-neuronal metal content and the expression of proteins related to metal homeostasis in the substantia nigra (SN) from patients with Parkinson's disease (PD), multiple sclerosis (MS), and control subjects. Metal quantification was performed via ion-beam micro-analysis in neuromelanin-positive neurons and the surrounding tissue. For proteomic analysis, SN tissue lysates were analyzed on a nanoflow chromatography system hyphenated to a hybrid triple-quadrupole time-of-flight mass spectrometer. We found increased amounts of iron in neuromelanin-positive neurons and surrounding tissue in patients with PD and MS compared to controls (4- to 5-fold higher) that, however, also showed large inter-individual variations. Copper content was systematically lower (-2.4-fold) in neuromelanin-positive neurons of PD patients compared with controls, whereas it remained unchanged in MS. Protein-protein interaction (PPI) network analyses revealed clusters related to Fe and Cu homeostasis among PD-deregulated proteins. An enrichment for the term "metal homeostasis" was observed for MS-deregulated proteins. Important deregulated hub proteins included hemopexin and transferrin in PD, and calreticulin and ferredoxin reductase in MS. Our findings show that PD and MS share commonalities in terms of iron accumulation in the SN. Concomitant proteomics experiments revealed PPI networks related to metal homeostasis, substantiating the results of metal quantification.

CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Enlighting Electron Routes In Oxyfunctionalizing Synechocystis sp. PCC 6803.

Phototrophic microorganisms, like cyanobacteria, are gaining attention as host organisms for biocatalytic processes with light as energy source and water as electron source. Redox enzymes, especially oxygenases, can profit from in-situ supply of co-substrates, i. e., reduction equivalents and O2 , by the photosynthetic light reaction. The electron transfer downstream of PS I to heterologous electron consuming enzymes in principle can involve NADPH, NADH, and/or ferredoxin, whereas most direct and efficient transfer is desirable. Here, we use the model organism Synechocystis sp. PCC 6803 to investigate, to what extent host and/or heterologous constituents are involved in electron transfer to a heterologous cytochrome P450 monooxygenase from Acidovorax sp. CHX100. Interestingly, in this highly active light-fueled cycloalkane hydroxylating biocatalyst, host-intrinsic enzymes were found capable of completely substituting the function of the Acidovorax ferredoxin reductase. To a certain extent (20 %), this also was true for the Acidovorax ferredoxin. These results indicate the presence of a versatile set of electron carriers in cyanobacteria, enabling efficient and direct coupling of electron consuming reactions to photosynthetic water oxidation. This will both simplify and promote the use of phototrophic microorganisms for sustainable production processes.

An Enzymatic Cofactor Regeneration System for the in-Vitro Reduction of Isolated C=C Bonds by Geranylgeranyl Reductases.

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.

SAT264 Effects Of Coenzymes In Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Model Mice

Abstract Disclosure: Y. Naiki: None. M. Miyado: None. R. Horikawa: None. N. Katsumata: None. S. Takada: None. M. Fukami: None. Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s and they need coenzymes respectively. Microsomal P450s include 21-hydroxylase need P450 oxidoreductase which coded by POR and ferredoxin and ferredoxin reductase, which coded by FDX and FDXR, respectively, work for mitochondrial P450s include 11β-hydroxylase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, and 11β-hydroxylase deficiency (11β-OHD), which caused by mutated CYP11B1 are the top two cause of CAH. CAH are treated with oral steroid supplementation, but optimal control remains difficult. Thus, new therapeutic approaches are still needed. We succeeded gene therapy for model mice of CAH and iPS cell from CAH patients (1). In this study, we examined the effects of induction of coenzyme genes with an AAV vector into a model mouse. A 21OHD mouse were made by breeding with H-2aw18 haplotype mice and treated with subcutaneous corticosteroid injection until 3 weeks of age. A 11β-OHD mouse were made by using the CRISPR/Cas9 method. A type 9 AAV (AAV9) containing Cyp21a cDNA (AAV9-Cyp21a1) was constructed with pAAV-CMV-shuttle. AAV9 containing Por cDNA (AAV9-Por), Cyp11b1 cDNA (AAV9-Cyp11b1), Fdx (AAV9-Fdx), Fdxr (AAV9-Fdxr) were also made. An AAV9-Cyp21a1 and AAV9-Por were mixed and injected into limbs muscles of a 21-OHD mouse and AAV9-Cyp11b1 mixed with AAV9-Fdx and AAV9-Fdxr injected into limbs muscles of 11β-OHD mouse. Serum progesterone (P4) and deoxycorticosterone (DOC) concentrations for 21-OHD mouse and DOC and corticosterone (B) concentrations for 11β-OHD mouse were measured before and after injection. P4 and DOC concentrations were measured by LC-MS/MS. An AAV9-Cyp21a1 and AAV9-Por injected mouse showed improved steroid synthesis up to 7 months after AAV9 induction. Comparing with a mouse injected only AAV9-Cyp21a1, significant reduction of P4/DOC was observed after 4 weeks of injection but there was no difference in the effectiveness of Cyp21a1 and Por combined treatment at the end of observation. Instead of a 21-OHD mouse, a 11β-OHD mouse did not show significant improvement by AAV mixture injected. These results indicate that extra-adrenal induction of Cyp21a1 and Por may ameliorate steroid metabolism in 21-OHD model mice. Although this coenzyme combined therapy showed some effects, but no long-term improvement of the efficacy exhibited. Instead of microsomal P450s, extra-adrenal induction with coenzyme genes did not work enough for mitochondrial P450s defects. Coenzymes of P450s are necessary for their activity, but our results suggest that the coenzyme combined gene therapeutic strategies are needed to be improved. Reference: (1) Naiki et al., Hum Gene Ther. 2022 Aug;33(15-16):801-809. Presentation: Saturday, June 17, 2023

FDXR drives primary and endocrine-resistant tumor cell growth in ER+ breast cancer via CPT1A-mediated fatty acid oxidation.

The majority of breast cancers (BCs) expressing estrogen receptor (ER) have shown endocrine resistance. Our previous study demonstrated that ferredoxin reductase (FDXR) promoted mitochondrial function and ER+ breast tumorigenesis. But the underlying mechanism is not clear. Liquid chromatography (LC) tandem mass spectrometry (MS/MS)-based metabolite profiling was utilized to reveal the metabolites regulated by FDXR. RNA microarray was utilized to determine the potential downstream targets of FDXR. Seahorse XF24 analyzer was performed to analyze the FAO-mediated oxygen consumption rate (OCR). Q-PCR and western blotting assays were used to measure expression levels of FDXR and CPT1A. MTS, 2D colony formation and anchorage-independent growth assays were used to evaluate the effects of FDXR or drug treatments on tumor cell growth of primary or endocrine-resistant breast cancer cells. We found that depletion of FDXR inhibited fatty acid oxidation (FAO) by suppressing CPT1A expression. Endocrine treatment increased the expression levels of both FDXR and CPT1A. Further, we showed that depletion of FDXR or FAO inhibitor etomoxir treatment reduced primary and endocrine-resistant breast cancer cell growth. Therapeutically, combining endocrine therapy with FAO inhibitor etomoxir synergistically inhibits primary and endocrine-resistant breast cancer cell growth. We reveal that the FDXR-CPT1A-FAO signaling axis is essential for primary and endocrine-resistant breast cancer cell growth, thus providing a potential combinatory therapy against endocrine resistance in ER+ breast cancer.